The transfusion of red blood cells is the primary method presently available for providing blood to patients experiencing hemorrhage or undergoing invasive medical procedures such as surgery. However, the use of blood presents problems for both healthcare workers and patients. For example, blood products require special storage conditions (i.e., freezing temperature) which can impose a short shelf life, and therefore large amounts of stored blood are discarded. Blood shortages occur because the supply from donors is not always assured. Blood transfusion requires properly matched blood types to avoid antigenic response, and also presents the possibility of contamination by blood born pathogens such as the human immunodeficiency virus. Thus, a search for blood substitutes has been undertaken in order to provide an alternative to blood transfusion.
Free hemoglobin solutions have been investigated as potential blood substitute products because workers would not have to be concerned with antigenic response (which is caused by non-hemoglobin blood factors) and because it might be easier to store and have a longer shelf life. However, it is known that free hemoglobin converts from its natural tetrameric form to a dimeric form that is rapidly excreted by the kidney. Renal toxicity has also been observed. Further, stroma free (cell free) hemoglobin is known to have a low P.sub.50 and high oxygen binding affinity (and therefore slow release) because of the absence of 2,3-diphosphoglycerate. Thus, efforts to overcome these problems have been directed toward developing a modified hemoglobin product, or some form of synthetic red blood cell.
In an attempt to overcome problems associated with the use of cell-free hemoglobin solutions, many have sought to create a synthetic red blood cell with hemoglobin encapsulated therein. For example, liposome encapsulated hemoglobin formulations and methods for making them are known in the art: U.S. Pat. Nos. 4,776,991 and 4,911,929 (Farmer), 4,532,130 (Djordjevich) and 4,133,874 (Miller); the disclosures of which are incorporated by reference herein.
Liposomes are microscopic vesicles made from phospholipids, which form closed, fluid filled spheres when dispersed with aqueous solutions. Phospholipid molecules are polar, having a hydrophilic head and two hydrophobic tails consisting of long fatty acid chains. Thus, when a sufficient concentration of phospholipid molecules are present in aqueous solutions, the mils spontaneously associate to exclude water while the hydrophilic phosphate heads interact with water. The result is a spherical, bilayer membrane in which the fatty acid tails converge in the interior of the newly formed membrane, and the polar heads point in opposite directions toward an aqueous medium. These bilayer membranes thus form closed, hollow spheres known as liposomes. The polar heads at the inner surface of the membrane point toward the aqueous interior of the liposome and, at the opposite surface of the spherical membrane, the polar heads interact with the surrounding aqueous medium. As the liposomes are formed, water soluble molecules can be incorporated into the aqueous interior, and lipophilic molecules may be incorporated into the lipid bilayer. Liposomes may be either multilamellar, like an onion with liquid separating many lipid bilayers, or unilamellar, with a single bilayer surrounding an aqueous center.
Methods for producing liposomes are well known in the art, and there are many types of liposome preparation techniques which may be employed to produce various types of liposomes. These can be selected depending on the use, the chemical intended to be entrapped, and the type of lipids used to form the bilayer membrane. The requirements which must be considered in producing a liposome preparation are similar to those of other controlled release mechanisms. They are: (1) a high percent of chemical entrapment; (2) increased chemical stability; (3) low drug toxicity; (4) rapid method of production; and (5) a reproducible size distribution.
The first method described to encapsulate drugs or other chemicals in liposomes involved the production of multilamellar vesicles (MLVs). Liposomes can also be formed as unilamellar vesicles (UVs), which generally have a size less than 0.5 .mu.m (.mu.m, also referred to as "microns"). There are several techniques known in the art which are used to produce unilamellar liposomes.
Smaller unilamellar vesicles can be formed using a variety of techniques, such as applying a force sufficient to reduce the size of the liposomes and or produce smaller unilamellar vesicles. Such force can be produced by a variety of methods, including homogenization, sonication or extrusion (through filters) of MLVs. These methods results in dispersions of UVs having diameters of up to 0.2 .mu.m, which appear as clear or translucent suspensions. Other standard methods for the formation of liposomes are known in the art, for example, methods for the commercial production of liposomes include the homogenization procedure described in U.S. Pat. No. 4,753,788 to Gamble, a preferred technique, and the method described in U.S. Pat. No. 4,935,171 to Bracken, which are incorporated herein by reference.
Another method of making unilamellar vesicles is to dissolve phospholipids in ethanol and inject them into a buffer, whereby the lipids will spontaneously rearrange into unilamellar vesicles. This provides a simple method to produce UVs which have internal volumes similar to that of those produced by sonication (0.2-0.5 L/mol of lipid). Another common method for producing small UVs is the detergent removal technique. Phospholipids are solubilized in either ionic or non-ionic detergents such as cholates, Triton X-100, or n-alkylglucosides. The drug is then mixed with the solubilized lipid-detergent micelles. Detergent is then removed by one of several techniques: dialysis, gel filtration, affinity chromatography, centrifugation or ultrafiltration. The size distribution and entrapment efficiencies of the UVs produced this way will vary depending on the details of the technique used.
The therapeutic uses of liposomes include the delivery of drugs which are normally toxic in the free form. In the liposomal form the toxic drug may be directed away from the sensitive tissue and targeted to selected areas. Liposomes can also be used therapeutically to release drugs, over a prolonged period of time, reducing the frequency of administration. In addition, liposomes can provide a method for forming an aqueous dispersion of hydrophobic chugs for intravenous delivery.
When liposomes are used to target encapsulated drugs to selected host tissues, and away from sensitive tissues, several techniques can be employed. These procedures involve manipulating the size of the liposomes, their net surface charge as well as the route of administration. More specific manipulations have included labeling the liposomes with receptors or antibodies for particular sites in the body. The route of delivery of liposomes can also affect their distribution in the body. Passive delivery of liposomes involves the use of various routes of administration, e.g., intravenous, subcutaneous and topical. Each route produces differences in localization of the liposomes. Two methods used to actively direct the liposomes to selected target areas are binding either antibodies or specific receptor ligands to the surface of the liposomes. Antibodies are known to have a high specificity for their corresponding antigen and have been shown to be capable of being bound to the surface of liposomes, thus increasing the target specificity of the liposome encapsulated drug.
Since the chemical composition of many drugs precludes their intravenous administration, liposomes can be very useful in adapting these drugs for intravenous delivery. Furthermore, since liposomes are essentially hollow spheres made up of amphipathic molecules, they can entrap hydrophilic drugs in their aqueous interior space and hydrophobic molecules in their lipid bilayer. Unwanted molecules that remain in the dispersion external to the liposomes, such as unentrapped agents, are removed by column chromatography or ultrafiltration. Although methods for making liposomes are well known in the art; it is not always possible to determine a working formulation without experimentation.
One important characteristic of a regulatory approved parenteral product is that it be sterile. Terminal sterile filtration is preferred to aseptic processing for the generation of a sterile parenteral product, and has been found to be the most effective in terms of processing and liposome stability. The best method for terminal sterile filtration is the sequential filtration of a dispersion of liposomes through a 0.45 and 0.22 micron filtration system, and liposomes larger than 0.2 .mu.m or aggregations of smaller liposomes will obstruct and clog this filter system, as well as the ultrafiltration system employed to remove unentrapped components. The Farmer patents disclose the small scale filtration of a liposome encapsulated hemoglobin formulation dispersed in a hyperosmotic buffered saline solution through a 0.22 micron filter. Similarly, Djordjevich discloses a laboratory process for filtering liposome encapsulated hemoglobin dispersed in a saline solution through a 0.22 micron filter for purposes of sterilization.
Another important aspect of developing a liposome formulation is to achieve a unimodal or controlled particle size distribution of unilamellar liposomes having a median size less then 0.2 .mu.m. Controlling the particle size distribution provides not only for a sterile filterable product but it also provides other numerous processing and pharmacological benefits such as RES avoidance and longer circulation times.
It has been desideratum to provide a process for preparing dispersion of hemoglobin encapsulating unilamellar liposomes that have a unimodal size distribution that do not aggregate, thus are capable of being ultrafiltered and are sterile filterable.